Abstract Many mammalian organs (skin, stomach, intestines, colon, and eye) possess a source of adult stem cells that continually replenishes their rapidly self-renewing epithelial surface. The limbus contains a small subpopulation of rare limbal stem cells (LSC) that continually repopulates the corneal epithelium. Patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in conjunctivalization of the corneal stroma that triggers neovascularization, chronic inflammation, and ultimately blindness due to an irreversibly opaque cornea. Several approaches have been used to replace LSC by transplanting limbal tissue or ex vivo expanded limbal cells. These procedures have obtained some success, mainly using autologous limbal tissue from patients with unilateral LSCD. Patients with bilateral LSCD have no source of autologous LSC and much less success was observed with allogeneic limbal tissue transplants. However, success of all these procedures was severely limited by the inability to prospectively identify and purify LSC. This problem was recently addressed by the three PIs of this proposal, who discovered that the ABCB5 gene, a new member of the ATP-binding cassette (ABC) superfamily of active transporters, is expressed by stem cells of the limbus. Normal function of ABCB5+ LSC is required for corneal development and repair, through critical roles in stem cell maintenance and survival, and knockout mice that lack ABCB5 do not develop a fully differentiated mature corneal epithelium. Importantly, ABCB5 is a cell surface protein and specific monoclonal antibodies developed by the laboratories of Co-PIs Drs. M. Frank and N. Frank are capable of isolating pure ABCB5-positive cells from the limbus. Transplantation of purified human ABCB5+ (but not ABCB5-) LSC onto the corneal stroma of immunodeficient mice with induced LSCD restored the corneal epithelium, indicating that this purified LSC population has the potential to significantly improve therapy for corneal disease associated with LSCD. Our currently funded RO1 project builds upon these results to address the important challenges that prevent successful stem cell therapy for patients with unilateral or bilateral LSCD, with the overall hypothesis that ABCB5+ stem cells from the limbus can be isolated and expanded ex vivo as a source of stem cells to regenerate the corneal epithelium when transplanted to recipients with either a unilateral or bilateral LSCD. This supplemental grant application is based upon early results of this project demonstrating that human ABCB5+ LSC can be expanded in vitro for use as successful pure stem cells grafts in pre-clinical therapeutic transplantation models. With supplemental funding, we wish now to focus on furthering these results with a view towards advancing ABCB5+ LSC transplantation to clinical testing, through (1) conducting GMP-conforming pilot production of in vitro-expanded LSC for IND-enabling studies, and (2) using GMP-produced in vitro-expanded LSC for initial preclinical IND-enabling studies (pharmacology/efficacy and mechanism of action, pharmacokinetics/biodistribution, toxicology, and carcinogenicity).